Compounds that inhibit the binding of RAF-1 or 14-3-3 proteins to the beta chain of IL-2 receptor, and pharmaceutical compositions containing same

ABSTRACT

The invention relates to compounds, such as proteins, peptides and organic compounds, capable of blocking or inhibiting the binding interaction of Raf-1 or 14-3-3 proteins to the β chain of IL-2, and pharmaceutical compositions containing such compounds. In vitro assays for isolating, identifying and characterizing such compound capable of inhibiting interaction of Raf-1 or 14-3-3 proteins to IL-2β are also provided.

This application is a national stage filing under 35 USC 371 fromPCT/US97/08542, filed May 22, 1997. This application also claims thebenefit of U.S. Provisional Application No. 60/018,183, filed May 23,1996 now abandoned.

FIELD OF THE INVENTION

The present invention concerns compounds such as proteins, peptides andorganic compounds which are characterized by their ability to block theinteraction between Raf-1 protein and/or 14-3-3 proteins with theintracellular domain of the β chain of the interleukin-2 receptormolecule (IL-2Rβ), and thereby block the intracellular signaling processmediated by IL-2Rβ. The compounds of the invention are intended toinhibit the activity of IL-2 or IL-15 where desired, for example inautoimmune diseases in general, or graft-versus-host reactions inparticular. The present invention also concerns in vitro assays for theisolation, identification and characterization of the above compounds,as well as pharmaceutical compositions containing as active ingredientone or more compounds of the invention.

BACKGROUND OF THE INVENTION

Interleukin-2 (IL-2) is a T-cell derived factor that amplifies theresponse of T cells to any antigen by stimulating the growth of the Tcells. Thus, IL-2 is a critical T-cell growth factor which plays a majorrole in the proliferation of T cells that occurs subsequent to antigenactivation, this proliferation resulting in the amplification of thenumber of T cells responsive to any particular antigen. IL-15 cangenerally substitute for IL-2 to exert most, if not all, of theseactivities (Bamford et al., 1994).

The high affinity (Kd:10⁻¹¹M) IL-2 receptor (IL-2R) is composed of atleast three non-covalently associated IL-2 binding proteins: the lowaffinity (Kd:10⁻⁸M) p55 (α chain) and the intermediate affinity subunits(Kd:10⁻⁹M) p75 (β chain) and p64 (γ chain) (Smith, K. A., 1988;Waldmann, T. A., 1993). Proliferative signals for the T cells aredelivered through high affinity IL-2 receptors consisting of all threesubunits, but not via the low affinity site (Robb, R. J. et al., 1984;Siegal, J. P. et al., 1987; Hatakeyama, M. et al., 1989). IL-2Rα,IL-2Rβ, and IL-2Rγ chains have 13, 286 and 86 amino acidintracytoplasmic domains, respectively.

IL-15, a cytokine with many IL-2-like activities, also utilizes theIL-2Rβ as a part of its receptor complex (Giri et al., 1994). ThisIL-2Rβ dependent signaling process is fundamental to the cellulareffects induced by the binding of IL-2 to its receptor (IL-2R) as wellas the effects induced by the binding of IL-15 to its receptor. TheIL-2Rβ and γ chains, but not the α chain, are essential for IL-2- aswell as IL-15-mediated signal transduction (Nakamura, Y. et al., 1994).The 64 kDa IL-2Rγ chain protein is rapidly phosphorylated on tyrosineresidues after stimulation with IL-2. The γ chain has also been shown tobe a part of other receptor complexes such as the receptor for IL-4 andIL-7 (Noguchi, M. et al., 1993; Russell, S. M. et al., 1993). Absence ofthe γ chain leads to a severe combined immunodeficiency disease inhumans (Noguchi, M. et al., 1993). IL-2Rγ contains sequences frompositions 288 to 321 homologous to the Src homology region 2 (SH2) thatcan bind to phosphotyrosine residues of some phosphoproteins. Anothermolecule, designated pp97, has been suggested to be the tyrosine kinasephysically associated with the IL-2Rγ chain (Michiel, D. F. et al.,1991).

An analysis of cells transformed with a series of IL-2Rβ chain deletionmutants identified a 46 amino acid serine and proline richintracytoplasmic region of the IL-2Rβ chain (a.a. 267-312), which iscrucial for growth promoting signal transduction (Hatakeyama, M. et al.,1989). This same region is crucial for promoting IL-15 mediated effects.Upon stimulation with IL-2, enzymatically active protein tyrosinekinases and, as the laboratory of the present inventors has previouslyshown (Remillard, B. et al., 1991), the novel lipid kinase,phosphatidyinositol-3-kinase activity blocks proliferation. Cells thatexpress wild-type IL-2Rα and γ chains and mutant IL-2Rβ chains lackingthis 46 a.a. region bind and internalize IL-2, but fail to proliferatein response to IL-2 (Hatakeyama, M. et al., 1989). An identical set ofcircumstances pertains to IL-15 responses. Although the intracytoplasmicdomain of the IL-2Rβ and γ chains lacks a protein tyrosine kinaseconsensus sequence, several cellular proteins are phosphorylated upontyrosine residues following IL-2 stimulation (Benedict, S. H. et al.,1987; Ferris, D. K. et al., 1989; Saltzmann, E. M. et al., 1988; Asao,H. et al., 1990; Mills, G. B. et al., 1990; Merida, I. and Gaulton, G.N., 1990). IL-2 induced protein tyrosine kinase activity is due, atleast in part, to activation of the p56^(lck) (lck), a src-familyprotein tyrosine kinase. Controversy exists as to whether theserine/proline rich (Fung, M. R. et al., 1991) or an adjacent tyrosinerich “acidic” region (Hatakeyama, M. et al., 1991) of the IL-2Rβ chainis the lck binding site.

IL-2 also stimulates phosphorylation on serine residues of severalproteins (Turner, B. et al., 1991; Valentine, M. V. et al., 1991).Raf-1, a serine/threonine kinase, has been identified as a likely signaltransducing element for several growth factor receptors (Carroll, M. P.et al., 1990; Morrison, D. K. et al., 1988; Baccarini, M. et al., 1991;Kovacina, K. S. et al., 1990; Blackshear, P. J. et al., 1990; App, H. etal., 1991). The Raf-1 molecule has a molecular weight of 74 kD and canbe divided into 2 functional domains, the amino-terminal regulatory halfand the carboxy-terminal kinase domains (for review see Heidecker, G. etal., 1991). Raf-1 has been identified as a crucial signal transducingelement for ligand activated EPO receptors (Carroll, M. P. et al.,1991). The IL-2Rβ chain and EPO receptors belong to the same family ofreceptors and share homologies within their cytoplasmic domains(D'Andrea, A. D. et al., 1989). Stimulation of the IL-2R results in thephosphorylation and activation of cytosolic Raf-1 serine/threoninekinase. IL-2R stimulation leads to a 5 to 10 fold immediate/earlyinduction of the c-raf-1 mRNA expression on freshly isolated, resting Tcells (Zmuidzinas, A. et al., 1991) and results in up to a 12-foldincrease in Raf-1 protein expression. In addition, a rapid increase inthe phosphorylation state of a subpopulation of Raf-1 moleculesprogressively increases through G1.

Enzymatically active Raf-1 appears in the cytosol of IL-2 stimulatedCTLL-2 cells (Hatakeyama, M. et al., 1991) and human T blasts(Zmuidzinas, A. et al., 1991). Following IL-2 stimulation, cytosolicRaf-1 molecules are phosphorylated on tyrosine and serine residues(Turner, B. et al., 1991). The laboratory of the present inventors havestudied the signaling pathway by which IL-2 signals T cells to begindividing. In these studies Raf-1 was identified in immunoprecipitates ofthe IL-2Rβ chain, suggesting that Raf-1 may be involved as an importantelement in IL-2 signaling. Further, it was determined that prior to IL-2stimulation, enzymatically active Raf-1 molecules are physicallyassociated with the IL-2Rβ chain and that following stimulation withIL-2, a protein tyrosine kinase phosphorylates Raf-1 thereby leading totranslocation of Raf-1 from the IL-2 receptor into the cytosol(Maslinski, W. et al., 1992). Moreover, dissociation of enzymaticallyactive Raf-1 from the IL-2Rβ chain, but not maintenance of IL-2Rassociated kinase activity, is completely abolished by genistein, apotent tyrosine kinase inhibitor (Maslinski, W. et al., 1992). Theabove-noted suggested requirement of Raf-1 for IL-2 signaling has beensupported by evidence showing that by blocking Raf-1 expression, IL-2could not induce T cell proliferation in the absence of Raf-1. Thus,from the afore-mentioned, it is widely accepted that activation of theRaf-1 serine/theonine kinase is critical for IL-2-mediated T-cellproliferation (see also Riedel et al., 1993).

Prior to IL-2 stimulation, several serine, but not tyrosine northreonine, residues of the IL-2Rβ chain are phosphorylated (Asao, H. etal., 1990). IL-2 induces rapid (i.e., within 10-30 min) phosphorylationof additional serines, tyrosines and threonines (Asao, H. et al., 1990;Hatakeyama, M. et al., 1991). Tyr 355 and Tyr 358 are major, but notexclusive, tyrosine phosphorylation sites of IL-2R (catalyzed byp561^(lck) in Vitro (Hatakeyama, M. et al., 1991)). The phosphorylationsites of the IL-2Rβ chain may play an important role in IL-2Rβ chainsignal transduction and interactions with accessory molecules (likep561^(lck) and Raf-1).

Phosphorylation of Raf-1 has also been demonstrated in a human T cellline following CD4 cross-linking. Activation of Raf-1 has also beenobserved following TCR/CD3 complex stimulation by CD3 or Thy 1cross-linking as well as an approximately four fold increase in c-raf-1mRNA. In this case, Raf-1 phosphorylation occurs only on serines and isnot observed if PKC had been down regulated. It is interesting to notein this context that GTPase-activating protein (GAP) activation and,consequently, Ras induction following TCR stimulation is also PKCmediated (Downward, J. et al., 1990).

However, the precise residues that form the contact points of p56^(lck)tyrosine kinase, and PI-3-kinase to the IL-2Rβ chain have not beenestablished. Indeed, two groups (Greene and Taniguchi) have utilizedgrossly truncated IL-2Rβ cDNA transfectants to analyze the binding sitesof the IL-2R to lck (Hatakeyama, M. et al., 1991; the Greene group;Turner, B. et al., 1991; the Taniguchi group). Although they usedessentially the same techniques and reagents, the conclusions of thesestudies are conflicting. It is possible that the use of drasticallytruncated mutants may result in conformational changes in the expressedprotein that confound attempts to precisely map the residue to residuecontact points required for ligand to ligand interaction. Moreover,recent data from Greene's group is more in line with Tanaguchi's data(Williamson, P. et al., 1994). However, the model cell line used by bothlaboratories (Baf/3) has been shown to signal differently than a T cellline CTLL2 (Nelson, B. H. et al., 1994). Thus, it is not completelyclear which portions of the IL-2Rβ chain are of most importance tonormal T cells.

The recent characterization of so-called “knockout” mice for IL-2 (i.e.,mice which lack IL2) has shown that about 50% die by nine weeks of age(Schorle, H. et al., 1991). Although these mice appear to bephenotypically normal and can mount some cell-mediated responses(Kundig, T. M. et al., 1993), they ultimately develop inflammatorydisease. Recently, it has been suggested that the reason the mice arestill relatively normal is that there is an additional cytokine (IL-15)that signals through the IL-2 receptor β and γ chains. Thus, there maybe some compensation by IL-15 in these mice for the lack of the IL-2molecules. On the other hand, deficiency of the IL-2Rγ chain in humansleads to a severe combined immunodeficiency, characterized by the nearabsence of both mature and immature T cells (Noguchi, M. et al., 1993).Further support for the importance of IL-2 in vivo comes from studiesutilizing anti-IL-2 antibodies. Marked immunosuppressive effects in bothtransplantation and autoimmune models have been obtained by usinganti-IL-2Rα monoclonal antibodies (Strom, T. B. et al., 1993). Clinicalefforts with similar anti-human IL-2Rα antibodies (produced in mice asmonoclonal antibodies) showed some efficacy but this was limited by arapid immune response in the human patients to the murine monoclonalantibody, i.e., human-anti-mouse antibodies (HAMA) were produced in thepatients a short time after treatment with the mouse-anti-human IL-2Rαmonoclonal antibodies.

Members of the highly conserved 14-3-3 protein family, first identifiedas abundant 27-30 kD acidic proteins in brain tissue (Moore et al.,1967) and later found in a broad range of tissues and organisms (Aitkenet al., 1992), were recently found to be associated with the products ofproto-oncogenes and oncogenes, such as Raf-1, Bcr-Ab1, and thepolyomavirus middle tumor antigen MT (Fu et al., 1994; Reuther et al.,1994; Pallas et al., 1994; Irie et al., 1994; Freed et al., 1994).14-3-3 appears to associate and interact with Raf-1 at multiple sites,i.e., amino terminal regulatory regions of Raf-1, kinase domain ofRaf-1, zinc finger-like region of Raf-1, etc., with primary sites ofinteraction located in the amino-terminal regulatory domain (Fu et al.,1994; Freed et al., 1994). In comparing sequences of Bcr, Bcr-Ab1 and MTat sites of interaction with 14-3-3, cysteine- and serine-rich regionswere found to be common elements and may be some of the determinantsresponsible for 14-3-3 binding (Morrison, 1994).

The results reported by Freed et al. (1994) and Irie et al. (1994)suggest that 14-3-3 modulates Raf-1 activity in yeast. For instance,Freed et al. (1994) found that over-expression of mammalian 14-3-3proteins in yeast stimulated the biological activity of mammalian Raf-1,and observed that mammalian Raf-1 immunoprecipitated from yeast strainsoverexpressing 14-3-3 had three- to four-fold more enzymatic activitythan Raf-1 from yeast strains lacking 14-3-3 expression. However, 14-3-3proteins alone are not sufficient to activate the kinase activity ofRaf-1, suggesting that 14-3-3 may be a cofactor involved in Raf-1activation (Morrison, 1994; Freed et al., 1994). Because 14-3-3constitutively associates with Raf-1 in vivo regardless of subcellularlocation or Raf-1 activation state or whether Raf-1 is bound to Ras (Fuet al., 1994; Freed et al., 1994), it is suggested that an alternatefunction of 14-3-3 may be a structural role in stabilizing the activityor conformation of signaling proteins (Morrison, 1994).

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

In view of the above-mentioned differences of the prior art, one of theaims of the present invention has been to determine the nature ofinteraction between the IL-2Rβ chain and Raf-1 and possibly otherproteins or peptides involved in the IL-2- or IL-15-mediatedintracellular processes. Accordingly, another aim of the presentinvention has been to find ways of inhibiting the binding between Raf-1and IL-2Rβ, and between IL-2Rβ, 14-3-3 and other proteins directlyinvolved in IL-2- or IL-15-mediated intracellular processes, and therebyprovide a way in which autoimmune diseases in general, all graftrejection and graft-versus-host reactions may be treated successfully.

The present invention is based on the development of in vitro assaysystems to determine the nature and specificity of the binding betweenRaf-1 and IL-2Rβ chain intracellular domain and the finding that theacidic region of the IL-2Rβ chain is essential for binding of Raf-1 toIL-2Rβ. The binding of IL-2Rβ to Raf-1 is an essential step in theintracellular signaling process mediated by the IL-2R and IL-15Rfollowing IL-2/IL-15 stimulation, and is implicated, amongst others, inautoimmune diseases in general, allograft rejection andgraft-versus-host reactions in particular.

More specifically, in accordance with the present invention it has nowbeen found that the intracellular domain of the IL-2Rβ chain directlybinds to Raf-1 and so-called 14-3-3 proteins. The acidic domain of theintracellular domain of the IL-2Rβ chain, that is homologous to the Raseffector domain, is critical for Raf-1 binding while the C-terminalportion of the intracellular domain of the IL-2Rβ chain interacts with14-3-3 protein. Further, the Raf-1 and 14-3-3 proteins form complexes onthe IL-2Rβ chain intracellular domain and in the presence ofenzymatically active p56^(lck), but not p59^(fYn), Raf-1/14-3-3complexes dissociate from the intracellular domain of the IL-2Rβ chain.Thus, the direct binding of Raf-1/14-3-3 proteins to the intracellulardomain of the IL-2Rβ chain by-passes the requirement for membranelocalization through activated Ras in other systems.

In view of the above, it thus arises that the co-localization of bothRaf-1 together with 14-3-3 on the acid domain and the C-terminal portionof the intracytoplasmic segment of the IL-2Rβ chain is an important stepin the intracellular signal transduction process mediated by the IL-2Rβchain. This interaction is therefore the target for the desiredcompounds which can disrupt or inhibit this interaction in accordancewith the present invention. Such disruption or inhibition of the aboveinteraction provides a specific inhibition of the IL-2/IL-15 initiatedintracellular signalling via the IL-2Rβ. Such inhibition is desirable inthe treatment of autoimmune diseases in general and graft-versus-hostreactions, in particular.

Accordingly, the present invention provides a compound capable ofbinding to Raf-1 protein, 14-3-3 proteins, or to the intracellulardomain of the IL-2Rβ chain and being able to inhibit the binding ofRaf-1 and/or 14-3-3 proteins to IL-2Rβ.

Embodiments of this aspect of the invention include: (i) A compoundselected from proteins, peptides and analogs or derivatives thereof, andorganic compounds; (ii) a compound being the 27 amino acid peptidecorresponding to amino acid resides 370 to 396 of SEQ ID NO:2, derivedfrom the acidic region of the mature human IL-2Rβ chain as set forth inFIG. 12 or analogs or derivatives thereof; (iii) a compound beingselected from analogs of said 27 amino acid peptide in which one or moreamino acid residues have been added, deleted or replaced, said analogsbeing capable of inhibiting the binding between Raf-1 and/or 14-3-3 andIL-2Rβ.

The present invention also provides a pharmaceutical compositioncomprising a compound of the invention or a mixture of two or morethereof, as active ingredient and a pharmaceutically acceptable carrier,excipient or diluent.

Further, the present invention provides an in vitro screening assay forisolating, identifying and characterizing compounds according to theinvention, capable of binding to Raf-1, 14-3-3 proteins, or IL-2Rβ chainintracellular domain, comprising (a) providing a synthetically produced,a bacterially produced or a mammalian cell produced protein selectedfrom IL-2Rβ chain protein or Raf-1 protein or 14-3-3 protein or portionsof any one thereof, or mixtures of any of the foregoing; (b) contactingsaid protein of (a) with a test sample selected from prokaryotic oreukaryotic cell lysates, a solution containing naturally derived orchemically synthetized peptides, or a solution containing chemicallysynthetized organic compounds, to form a complex between said proteinand said test sample; (c) isolating the complexes formed in (b); (d)separating the test sample from the protein in the complexes isolated in(c); and (e) analyzing said separated test sample of (d) to determineits nature. An embodiment of the above assay is an in vitro screeningassay for isolating, identifying and characterizing compounds capable ofbinding to Raf-1, 14-3-3 proteins or IL-2Rβ chain intracellular domain,as described in Examples 1-6 herein.

Other embodiments of the above screening assay of the invention includean in vitro screening assay wherein said assay is the herein describedcell-free assay system; an in vitro screening assay wherein said assayis the herein described totally cell-free assay system; an in vitroassay for screening a compound capable of binding to Raf-1, and/or14-3-3 proteins or IL-2Rβ intracellular domain and inhibiting thebinding between Raf-1 and IL-2Rβ, said assay comprising the steps ofdetermining the protein kinase reaction as described herein in Examples1-6; as well as compounds isolated, identified and characterized by thein vitro assays according to the invention.

Accordingly, the present invention also provides: (i) compoundsisolated, identified and characterized by any of the above in vitroassays; (ii) a pharmaceutical composition for the treatment ofautoimmune diseases or graft-versus-host reactions containing a compoundof the invention; and (iii) use of a compound of the invention for thetreatment of autoimmune diseases transplant rejection orgraft-versus-host reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically the structure of the IL-2Rβ fusion proteinsas described in Example 1;

FIG. 2 depicts the results illustrating the binding of Raf-1 from T-celllysates to FLAG-HMK-IL-2Rβ chain related proteins as described inExample 1;

FIG. 3 depicts the results illustrating the interaction betweenbacterially derived (His)₆-Raf-1 proteins with FLAG-HMK-IL-2Rβ chainrelated proteins as described in Example 2;

FIG. 4 depicts the results illustrating the products of protein kinasereaction performed on anti-FLAG beads coated with FLAG-HMK-IL-2Rβ chainproteins and exposed to T-cell lysates as described in Example 3;

FIG. 5 depicts the results illustrating the products of serine/threoninekinase reaction performed on anti-FLAG beads coated with FLAG-HMK-IL-2Rβchain and exposed to T-cell lysates, as described in Example 3;

FIG. 6 depicts the results illustrating the products of protein kinasereaction performed on anti-FLAG beads coated with FLAG-HMK-IL-2Rβ chainrelated proteins and exposed to T-cell lysates, as described in Example3;

FIGS. 7(a-c) depict schematically the structure of the IL-2Rβ fusionproteins prepared for expression in mammalian (COS) cells (FIG. 7a,IL-2Rβ chain contructs) and in bacterial cells (FIG. 7b, FLAG-HMK-IL-2Rβchain constructs), as well as the results of expression of these fusionproteins (FIG. 7c), as described in Example 4;

FIGS. 8(a-c) depict the results illustrating the direct interactionbetween Raf-1 and 14-3-3 proteins with IL-2Rβ chain or portions thereof,as described in Example 4;

FIG. 9 depicts a schematic representation of the homology between IL-2Rβchain (human) (amino acid residues 372 to 396 of SEQ ID NO:2) and theRas (human) protein (SEQ ID NO:3), as described in Example 4;

FIGS. 10(a-b) depict the results illustrating the abrogation byenzymatically active p56^(lck) of Raf-1 and 14-3-3 binding to the IL-2Rβchain, as described in Example 4;

FIG. 10c depicts the results illustrating the binding of Raf-1 and14-3-3 proteins from T-cell lysates to the IL-2Rβ chain as described inExample 4.

FIG. 11 is a schematic illustration of the determination of theRaf-1/IL-2Rβ chain contact points as described in Example 5; and

FIG. 12 is a schematic representation of the amino acid sequence of thehuman IL-2Rβ chain (SEQ ID NO:2), as described in Example 5. Theextracytoplasmic domain is in the upper part of the figure in upper caseletters. The peptide leader is indicated by lower case letters and thetransmembrane region by underlined letters. The acidic region (aa313-382) is indicated by dashed underlined letters and the putativeregion (aa 345-371) involved in IL-2Rβ/Raf-1 interaction is shown byitalic letters.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail in thefollowing non-limiting examples and accompanying figures:

EXAMPLE 1 IL-2Rβ Chain Interaction with Raf-1 Proteins: the IL-2Rβ ChainRegion Involved in Raf-1 Binding

As mentioned hereinabove, the direct interaction of the IL-2Rβ chain andRaf-1 binding has not been previously described. It has been widelybelieved that the IL-2Rβ mediated activation of Raf-1 involves theintermediacy of other proteins. In addition, it has not previously beendetermined whether or not 14-3-3 proteins ate capable of binding to theIL-2Rβ chain directly. Again, other intermediate proteins have beenimplicated in 14-3-3 binding. Furthermore, characterization of theproteins that are associated with the IL-2Rβ chain is limited by the lowcopy number of receptors per T cell (2-3×10³ receptors/cell), complexityof the interactions between the receptor protein and the myriad ofassociated proteins.

Accordingly, there has been developed in accordance with the presentinvention, a cell-free system in order to analyze the interactionbetween the IL-2Rβ chain and Raf-1 and/or 14-3-3 proteins, inparticular, to identify the region(s) of the IL-2Rβ chain essential forbinding to Raf-1 and/or 14-3-3 proteins. The binding of the 14-3-3proteins to IL-2Rβ is set forth in Example 4. This cell-free system wasinitially prepared as follows:

(i) The IL-2Rβ chain cytoplasmic domain was cloned in a bacterialexpression system and expressed as part of a fusion protein downstreamfrom 17 hydrophilic amino acids comprising an antigenic epitope (FLAG)and a recognition site for heart muscle kinase (HMK) that permits invitro radiolabeling of the fusion protein with [γ³²P]-ATP and HMK(LeClair, K. P. et al., 1992; Blanar, M. A. and Rutter, R. J., 1992).The FLAG-HMK-IL-2Rβ chain cytoplasmic domain expression plasmid wasconstructed by ligating the appropriate 1107 bp (NcoI-BamHI) cDNAfragment from the IL-2Rβ chain into the FLAG-HMK vector (LeClair et al.,1992; Blanar and Rutter, 1992) using synthetic linkers that facilitatedcloning and maintain the proper translational frame. BL-21 pLysSbacteria were transformed with the FLAG-HMK-IL-2Rβ construct, andprotein expression was induced as described (LeClair et al., 1992).

(ii) In order to study the interaction of IL-2Rβ chain withintracellular molecules, the FLAG-HMK-IL-2Rβ chain cytoplasmic domainfusion protein was purified from bacterial lysate using the M2 anti-FLAGmonoclonal antibody in a standard affinity chromatography procedure.More specifically, bacterial lysate proteins were absorbed ontoanti-FLAG (M2) affinity column (IBI-Kodak, New Haven, Conn., USA). Afterwashing the column, the adsorbed proteins were eluted with eitherglycine buffer (pH 3) or FLAG peptide (10⁻⁴M). Proteins in variousfractions were analyzed for expected size (about 33 kDa) and purityafter separation on SDS-PAGE and Commassie blue staining. The presenceof a functional HMK recognition site was confirmed by phosphorylation ofthe purified 33 kDa IL-2Rβ chain fusion protein by HMK. Morespecifically, the eluted fusion proteins were tested for susceptibilityto phosphorylation by incubation with the catalytic subunit of bovineheart muscle kinase (Sigma) (1 U/ul) in buffer containing 20 mMTric-HCl, pH 7.5, 1 mM DTT, 100 mM NaCl, 10 mM MgCl₂ and 1 μCi [(γ³²P]ATP for 30 min at 37° C. followed by SDS-PAGE and autoradiography.Purified FLAG-HMK-IL-2Rβ chain fusion proteins were used as an affinityreagent to probe for cytosolic proteins present in lysates of human Tcells, metabolically labeled with [³⁵S]-methionine, that bind to theIL-2Rβ chain. The human T cells being peripheral blood mononuclear cellswere isolated using Ficoll-Hypaque, stimulated with phytohemagglutinin(5 μg/ml) in culture for 72 h, washed, maintained in culture for 3 daysin the presence of IL-2 (10 U/ml), and then incubated without IL-2 for24 hours. For [³⁵S]-methionine labeling, the cells were suspended at4×10⁷ cells/ml at 37° C. followed by addition of 0.5 mCi of[³⁵S]-methionine for 4 hours prior to lysis in Dounce homogenizationbuffer and application of the lysate to the affinity column. Several[³⁵S]-labeled proteins were retained by the FLAG-HMK-IL-2Rβ chaincytosolic domain fusion protein bound to the affinity column. One ofthese proteins was identified as Raf-1 by immunoblotting using apolyclonal antibody specific for the SP-63 peptide which corresponds tothe C-terminal fragment of Raf-1. A molar excess of the competing SP-63peptide blocked binding of the anti-SP-63 antibody to Raf-1.

(iii) In order to test for serine/threonine kinase activity, proteinseluted from FLAG-HMK or FLAG-HMK-IL-2Rβ chain affinity columns by FLAGpeptide were diluted 1:1 in kinase buffer (25 mM HEPES, pH 7.5, 10 mMMgCl₂, 1 mM DTT) with or without genistein (10 μg/ml) and Histone Hi (20μg/ml) was added. The kinase reaction was initiated by the addition of 1μCi of [γ³²P]-ATP and 25 μM ATP. After 30 min at 24° C., the reactionwas stopped by addition of reducing SDS-PAGE sample buffer and boiling.The results showed that there was serine/threonine kinase activity amongthe human T-cell derived proteins bound to the IL-2Rβ chain affinitycolumn, and this protein kinase activity was not inhibited by treatmentwith the tyrosine kinase inhibitor, genistein. These results confirmedthat IL-2Rβ cytoplasmic domain chain fusion proteins can be used tostudy the binding of IL-2Rβ chain and cellular Raf-1 serine/threoninekinase in vitro, i.e., in a cell-free system.

Using the basic cell-free system described above, a number ofFLAG-HMK-IL-2Rβ chain wild type FLAG-HMK-IL2Rβ chain deletion mutantproteins were then studied with respect to this specific interactionwith Raf-1 proteins present in T-cell lysates. These FLAG-HMK-IL2Rβchain wild type (WT) and deletion mutants lacking certain defineddomains of the IL-2Rβ chain were used to identify the IL-2Rβ chaindomain involved in Raf-1 binding. Assay conditions were similar to thosedescribed above. Briefly, bacterially produced proteins: (a)FLAG-HMK-IL-2Rβ chain wild type (WT); (b) FLAG-HMK-IL-2Rβ chaincontaining only the proline rich C-terminal (CT⁺); FLAG-HMK-IL-2Rβ chainmutants lacking; (c) the serine rich region (S⁻); (d) the acidic domain(A⁻); (e) both acidic domain and proline rich C-terminal (A⁻CT⁻); or (f)FLAG-HMK vector (v) which does not contain IL-2Rβ chain sequences(negative control), were absorbed on anti-FLAG affinity beads andwashed. In FIG. 1, there is shown, schematically, all of the constructs,i.e., FLAG-HMK-IL-2Rβ chain fusion proteins produced in transformedbacterial cells and used in this study. These FLAG-HMK- fusionproteins-coated beads were then used as affinity reagents to absorbRaf-1 proteins present in T-cell lysates. T-cell derived proteins boundto FLAG-HMK fusion proteins were then eluted using buffer containingFLAG peptide, separated on SDS-PAGE, transferred onto Immobilon membraneand blotted with anti-Raf-1 antibody.

These experiments were repeated a number of times and the resultsindicated that: affinity beads coated with FLAG-HMK-IL-2Rβ chain WT orFLAG-HMK-IL-2Rβ chain S-mutant bind T-cell derived Raf-1 proteinsequally well; FLAG-HMK-IL-2Rβ chain mutant A⁻ proteins exhibitdiminished binding of Raf-1 proteins (50-80% decrease of Raf-1 bindingin comparison to WT control was observed); and there is no binding ofRaf-1 proteins to FLAG-HMK-IL-2Rβ chain mutants lacking both acidic andC-terminal domains (mutant A⁻CT⁻), FLAG-HMK-IL-2Rβ chain CT⁺ proteins(i.e. containing only the proline rich C-terminal) or FLAG-HMK vector(V) control. The results of one representative experiment is shown inFIG. 2, which is a reproduction of the relevant bands of an immunoblotof the above noted fusion proteins separated on SDS-PAGE, transferred tothe Immobilon membrane and blotted with the anti-Raf-1 antibody.Relative band intensity is apparent from the immunoblot, and thecalculated volume of each band corresponding to each different fusionprotein is indicated below the band.

EXAMPLE 2 The Interaction of (His)₆-Raf-1 Proteins with FLAG-HMK-IL-2RβChain WT and FLAG-HMK-IL-2Rβ Chain Deletion Mutant Proteins

In order to study direct interaction of the IL-2Rb chain and Raf-1proteins two Raf-1 related fusion proteins, i.e., FLAG-HMK-Raf-1 and(His)₆-Raf-1 proteins were constructed, bacterially expressed andpurified on affinity resins.

For the construction of the FLAG-HMK-Raf-1 expression plasmid, PCR wasperformed using the Raf-1 cDNA as template and oligonucleotide primersdesigned to facilitate cloning into the FLAG-HMK-vector (for FLAG-HMKvector, see Example 1). FLAG-HMK-Raf-1 protein was produced in BL-21pLysS bacteria by IPTG induction, and purified on anti-FLAG affinityresin. Affinity purification yielded a 72-74 kD protein which wasrecognized by anti-Raf-1 antibody.

For the construction of the (His)₆-Raf-1 expression plasmid, PCR wasperformed using the Raf-1 cDNA as template and oligonucleotide primersdesigned to facilitate cloning into the pQE-30 plasmid according to themanufacturer's protocol (QIAGEN, QIAexpressionist; Chatsworth, Calif.).(His)₆-Raf-1 protein was produced in M15 bacteria by IPTG induction, andpurified on Ni-NTA resin (QIAGEN). Affinity purification yielded a 72-74kD protein which was recognized by anti Raf-1 antibody.

On the basis of the above results, we then analyzed the specificrequirements of IL-2Rβ chain regions for binding to Raf-1. In thisanalysis the above noted cloned, i.e., bacterially produced Raf-1protein was utilized, the bacterially produced protein being a(His)₆-Raf-1 protein as a result of the cloning of the Raf-1 sequenceinto the expression vector. This has no effect on the Raf-1 activity.

The use of such a bacterially produced (His)₆-Raf-1 protein in thesebinding studies provides yet another advantage over the basic cell-freesystem in that a totally cell-free system is obtained, i.e., purifiedbacterially produced IL-2Rβ chain fusion proteins are reacted withpurified Raf-1 and not with Raf-1 within a T-cell lysate.

Accordingly, in this totally cell-free assay, FLAG-HMK-IL-2Rβ chain wildtype and deletion mutants lacking at least one of several defineddomains of the IL-2Rβ chain (see Example 1) were used to identify theIL-2Rβ chain domain involved in Raf-1 binding. Assay conditions weresimilar to those described in Example 1. Briefly, bacterially producedproteins: FLAG-HMK-IL-2Rβ chain wild type (WT), FLAG-HMK-IL-2Rβ chaincontaining only proline rich C-terminal (CT⁺), FLAG-HMK-IL-2Rβ chainmutants lacking:serine rich region (S⁻), acidic domain (A⁻), acidicdomain and proline rich C-terminal (A⁻CT⁻) were incubated withbacterially produced (His)₆-Raf-1 (for all constructs see FIG. 1)followed by adsorption of FLAG-HMK-IL-2Rβ chain/Raf-1 complexes onanti-FLAG affinity beads. After extensive washing, IL-2Rβ chain/Raf-1complexes were competitively eluted from anti-FLAG beads using buffercontaining FLAG peptide. Eluted proteins were separated on SDS-PAGE,transferred onto Immobilon membrane and blotted with anti-Raf-1antibody.

The results of one representative experiment is shown in FIG. 3, whichis a reproduction of the relevant bands of an immunoblot of the abovenoted proteins separated on SDS-PAGE, transferred to the Immobilonmembrane and blotted with the anti-Raf-1 antibody. Relative bandintensity is apparent from the immunoblot and the calculated volume ofeach band corresponding to each different fusion protein is indicatedbelow the band. It should be noted that in FIG. 3, the two extreme righthand samples, namely the second “WT” and the “VV” are positive andnegative controls (see below) and the “+” and “−” signs indicate whichIL-2Rβ-FLAG construct was reacted with Raf-1 protein. These experimentswere repeated a number of times, essentially with the same results:

(i) FLAG-HMK-IL-2Rβ chain (WT) and deletion mutant lacking the serinerich region (S⁻) of the IL-2Rβ chain bind Raf-1 proteins equally well.(ii) In contrast, mutants lacking the acidic domain of IL-2Rβ chain (A⁻)express a significantly reduced capacity to bind Raf-1. The amount ofRaf-1 proteins bound to FLAG-HMK-IL-2Rβ A⁻ mutant as estimated usingHewlett Packard ScanJet varied between 17% to 50% of the positivecontrol value, i.e., 17-50% of Raf-1 binding to FLAG-HMK-IL-2Rβ chainWT. (iii) Mutants lacking both acidic and C-terminal proline richdomains (A⁻CT⁻, also designated FLAG-HMK-IL-2Rβ S⁺, do not bind Raf-1proteins (0% of the positive control). (iv) FLAG-HMK-IL-2Rβ chain mutantcontaining only proline rich C-terminal (CT⁺) expressed (0%-10%) bindingto Raf-1 proteins. The two negative controls which were carried outwere:

1) lysates of bacteria transformed with FLAG-HMK vector (V) alone (noinsert) were incubated with equal amount of bacterial lysates containing(His)₆-Raf-1 proteins followed by adsorption of proteins onto anti-FLAGbeads, washing and elution with buffer containing FLAG peptide. Thiscontrol sample is at the extreme right hand side of FIG. 3 (“V”).

2) Bacterial lysates containing FLAG-HMK-IL-2Rβ chain WT proteins werealso incubated with equal amount of lysates prepared from bacteriatransformed with vector encoding (His)₆-proteins with no insert. Thiscontrol was undertaken to exclude the possibility that MIS bacteriacontain Raf-1-like proteins that may interact with the FLAG-HMK-IL-2Rβchain proteins. This control sample is second from the extreme righthand side of FIG. 3 (the second “WT”).

In view of the above results it is apparent that the acidic domain ofthe IL-2Rβ chain is required for optimal binding of Raf-1 proteins. Itis also possible that a portion of the proline-rich cytoplasmic tail isrequired for direct binding of Raf-1.

EXAMPLE 3 IL-2Rβ Chain Interaction with Raf-1 Proteins: Serine/ThreonineKinase Activity

The events that lead to activation of Raf-1 serine/threonine kinase inT-cells are unknown. Raf-1 possesses an N-terminal regulatory domain anda C-terminal catalytic domain which are separated by a serine-rich hingeregion. It is believed that the regulatory domain folds over the hingeregion onto the catalytic domain, thereby suppressing kinase activity(McGrew, B. R. et al., 1992; Bruder, J. T. et al., 1992; Stanton, V. P.et al., 1989). Consistent with this model, N-terminal truncated Raf-1proteins express constitutive kinase activity (Stanton, V. P. et al.,1989). Binding of the IL-2Rβ to the regulatory domain of Raf-1 mayactivate the kinase through a conformational change (Maslinski, W. etal., 1992). To determine whether direct binding of Raf-1 to the IL-2Rβchain induces activation of Raf-1 serine/threonine kinase activity, aFLAG-HMK-Raf-1 fusion protein was constructed and expressed.

In order to test whether direct interaction of IL-2Rβ chain cytoplasmicdomain and Raf-1 induces activation of Raf-1 kinase, we utilized astandard serine/threonine kinase assay (see references in Example 1 and2) to monitor kinase activity of Raf-1 alone and after interaction withthe IL-2Rβ chain fusion protein. Neither the purified FLAG-HMK-IL-2Rβcytoplasmic domain protein nor the FLAG-HMK-Raf-1 protein aloneexpressed serine/threonine kinase activity. Similarly, when bothproteins were combined in equimolar concentrations, serine/threoninekinase activity was not observed. These results indicate that (i) directinteraction of the IL-2Rβ chain and Raf-1 proteins is not sufficient toactivate enzymatic activity of Raf-1 and (ii) other factor present inT-cells may be required for mediating Raf-1 kinase activity. In order totest the later notion, using the above noted approach, serine/threoninekinase activity as a result of FLAG-HMK-IL-2Rβ chain interaction withT-cell derived proteins, which included Raf-1, was studied.

(a) Using the above noted approach, serine/threonine kinase activity asa result of FLAG-HMK-IL-2Rβ chain interaction with T-cell derivedproteins, which included Raf-1, was studied.

FLAG-HMK-IL-2Rβ chain wild type and deletional mutants lacking certaindefined domains of the IL-2Rβ chain (see Examples 1 and 2 and FIG. 1)were used to identify IL-2Rβ chain domain involved in binding T-cellderived, active serine/threonine kinase. Assay conditions were similarto those noted above. Briefly, bacterially produced proteins:FLAG-HMK-IL-2Rβ chain wild type (WT), FLAG-HMK-IL-2Rβ chain containingonly proline rich C-terminal (CT⁺), FLAG-HMK-IL-2Rβ chain mutantslacking:serine rich region (S⁻), acidic domain (A⁻), both acidic domainand proline rich C-terminal (A⁻CT⁻), or FLAG-HMK vector which does notcontain IL-2Rβ chain sequences (negative control) (for all constructssee diagram on FIG. 1) were absorbed on anti-FLAG affinity beads andwashed. FLAG-HMK-fusion proteins coated beads were further used asaffinity reagents to absorb proteins present in T-cell lysates. T-cellderived proteins bound to FLAG-MHK fusion proteins were then tested forserine/threonine kinase activity in the absence or presence of exogenoussubstrates: Histone H-1 or (His)₆-Mek-1. Products of kinase reactionswere boiled in SDS-PAGE sample buffer followed by separation onSDS-PAGE, transfer onto Immobilon membrane and autoradiography.

The following are the experiments that were carried out and theirresults:

(i) Kinase reaction performed in the absence of exogenously addedsubstrate. Affinity beads coated with FLAG-HMK-IL-2Rβ chain (WT) orFLAG-HMK-IL-2Rβ chain S⁻-mutant (S⁻) bind T-cell derived protein(s)expressing serine/threonine kinase activity as reflected byphosphorylation of p70 protein. This protein may be Raf-1 insofar as itcomigrates with Raf-1 protein. In contrast, there is no phosphorylatedband p70 in T-cell lysates retained on beads coated with otherFLAG-MHK-IL-2Rβ chain related fusion proteins (mutants A⁻, A⁻CT⁻, CT⁺)or bacterial lysates containing vector (V) control. These experimentswere repeated a number of times with essentially the same results. Theresults of one representative experiment is shown in FIG. 4, which is areproduction of an autoradiogram of the products of the protein kinasereaction performed on anti-FLAG beads coated with the various IL-2Rβfusion products, incubated with T cell lysates and subsequentlysubjected to SDS-PAGE and autoradiography.

(ii) Kinase reaction Performed in the presence of Histone-H-1. There isan increased, genistein (tyrosine kinase inhibitor) -independentphosphorylation of Histone-H-1 in T-cell lysates retained on affinitybeads coated with FLAG-HMK-IL-2Rβ chain. Control affinity beads coatedwith proteins isolated from bacteria transformed with vector alone andexposed to T-cell lysates retain only background level ofserine/threonine kinase activity. These experiments were repeated anumber of times. The results of a representative experiment are shown inFIG. 5 which is a reproduction of an autoradiogram of the products ofthe kinase reaction performed, in the presence of Histone H-1, onanti-FLAG beads coated with the IL-2Rβ chain construct (WT) and exposedto T-cell lysates in the presence of genistein (lane 3), or in theabsence of genistein (lane 2) and then subjected to SDS-PAGE andautoradiography. The control (lane 1) was carried out with the FLAG-HMKvector alone (no insert).

(iii) Kinase reaction performed in the presence of kinase defective(His)₆-Mek-1 proteins. An increase of the level of (His)₆-Mek-1 kinasephosphorylation was observed in the presence of anti-FLAG beads coatedwith FLAG-HMK-IL-2Rβ chain WT and S⁻ and exposed to T-cell lysates.Background levels of (His)6-Mek-1 kinase phosphorylation were observedin the presence of anti-FLAG beads coated with other FLAG-MHK-IL-2Rβchain related mutants (mutants A⁻, A⁻CT⁻, CT⁺) or bacterial lysatescontaining vector control. The results of a representative experimentare shown in FIG. 6 which is a reproduction of an autoradiogram of theproducts of the kinase reaction performed, in the presence of(His)₆-Mek-1 proteins, on various IL-2Rβ chain constructs exposed toT-cell lysates and then subjected to SDS-PAGE and autoradiography.

From the results shown in FIGS. 4-6, it is apparent that anti-FLAGaffinity beads coated with FLAG-HMK-IL-2Rβ chain wild type (WT) orFLAG-HMK-IL-2Rβ chain mutant lacking serine-rich region (mutant S⁻) andexposed to T-cell lysates, retain active serine/threonine kinase that(i) phosphorylates p70 band which comigrates with Raf-1 proteins, and(ii) phosphorylates kinase inactive (His)₆-Mek-1 proteins. In parallelexperiments, carried out in the presence of other FLAG-HMK-IL-2Rβ chainrelated proteins (mutants A⁻, A⁻CT⁻, CT⁺) or bacterial lysatescontaining vector control, these kinase activities are absent. Takentogether these results indicate that enzymatically activeserine/threonine kinase Raf-1 binds to the acidic region of the IL-2Rβchain.

(b) Following on the approach taken in (a) above, serine/threoninekinase activity as a result of FLAG-HMK-IL-2Rβ chain interaction withbacterially produced (His)₆-Raf-1 proteins, was studied. As noted inExample 2 above, this totally cell-free system has advantages over thecell-free system in (a) above in which T-lysates were used containingthe Raf-1 proteins.

Bacterial lysates containing FLAG-HMK-IL-2Rβ chain wild type and (His)₆-Raf-1 proteins were used (see Example 2) to test the hypothesis thatthe IL-2Rβ chain induces catalytic activity of Raf-1. Assay conditionswere similar to those described above. Briefly, bacterially producedproteins: FLAG-HMK-IL-2Rβ chain wild type (WT) or FLAG-HMK (negativecontrol) (for all constructs see FIG. 1) were incubated with bacteriallysates containing either (His)₆-Raf-1 or (His), (negative control)followed by the absorption of protein complexes on anti-FLAG affinitybeads. Washed beads were tested for the presence of serine/threoninekinase activity in the presence of the exogenously added substrate,enzymatically inactive (His)₆-Mek-1 kinase protein. Products of kinasereactions were boiled in SDS-PAGE sample buffer followed by separationon SDS-PAGE, transfer onto Immobilon membrane and autoradiography. Theseexperiments were repeated a number of times with similar results:interaction of FLAG-HMK-IL-2Rβ chain with (His)₆-Raf-1 proteins did notresult in the induction of kinase activity toward Mek-1 kinase.

These results therefore indicate the possibility that some other factor(or co-factor) is necessary for mediating the Raf-1 kinase activity,this being present in the T cell lysates (see (a) above) but not in themore purified (His)₆-Raf-1 preparation from transformed bacterial cells.It is possible that proteins of the 14-3-3 family are involved bybinding to Raf-1 and thereby mediate its activity. Such 14-3-3 familyproteins have recently been described (Freed et al., 1994; Irie et al.,1994; Morrison, 1994), and these have been studied as set forth inExample 4 below.

EXAMPLE 4 IL-2Rβ Chain Interaction with Raf-1 and/or 14-3-3 Proteins:the IL-2Rβ Chain Region Involved in Raf-1 and/or 14-3-3 Protein Binding

In another set of experiments to identify the IL-2Rβ chain domain(s)that might interact with Raf-1 and/or 14-3-3proteins, cDNAs encoding theIL-2Rβ chain or mutants lacking segments of its cytoplasmic domain wereprepared and expressed in COS cells.

(i) In these experiments (see also Example 1 a (i) and (ii) above) cDNAencoding human IL-2Rβ chain wild type (IL-2Rβ-WT) (Hatakeyama et al.,1989), was digested with Xba I and inserted into expression vectorpRcCMV (Invitrogene). A cDNA encoding mutant IL-2Rβ lacking 71 aminoacids (aa 252-322), that contain box 1 (Murakami et al, 1991) and serinerich region critical for signal transduction (Hatakeyama et al., 1989)IL-2Rβ-box 1⁻S⁻, was made by cloning the full length wild type IL-2Rβchain cDNA (SEQ ID NO:1) into the XbaI site of pBluescript II SK(Stratagene). This construct was then digested with NcoI-AflII. TheNcoI/AflII sites were ligated with double stranded linker composed ofoligonucleotides:

5′CATGGCTGAAGAAGGTC3′ (sense, bases 946-962; SEQ ID No:4) and

5′TTAAGACCTTCTTCAGC3′ (antisense, bases 950-962, plus an AflII site; SEQID No:5). This construct was then digested with XbaI and fragmentcontaining sequences encoding IL-2Rβ chain was cloned back into pRcCMV.For the construction of IL-2Rβ-A⁻ mutant, pRcCMV-IL-2Rβ was digestedwith XbaI and cloned into XbaI site of pTZ19R (Pharmacia). Thisconstruct was then digested with NcoI-BstXI. The 964 bp fragmentcontaining sequences encoding most of the cytoplasmic domain of theIL-2Rβ chain was replaced with a 754 bp fragment obtained from NcoI andBstXI digestion of the AR(DRI)59/60 plasmid (Le Clair et al., 1992;Blanar et al., 1992) containing FLAG-HMK-IL-2Rβ-A⁻ mutant encoding cDNA(see below). The resultant pTZ-IL-2Rβ-A⁻ plasmid contains sequencesencoding an IL-2Rβ chain but lacking 210 bases encoding acidic domainwas then digested with XbaI, and a fragment containing sequencesencoding IL-2Rβ-A⁻ was cloned back into pRcCMV.

For the construction of plasmid FLAG-HMK-IL-2Rβ chain cytoplasmic domainwild type (FLAG-HMK-IL-2Rβ-WT), a 1107 bp cDNA (see also (i) above) wasexcised from IL-2Rβ chain cDNA with NcoI-BamHI and ligated withsynthetic, in frame double stranded linker EcoRI/NcoI (made fromoligonucleotides: sense 5′AATTCAACTGCAGGAACACCGGGC3′ (EcoRI site plusbases 927-944; SEQ ID No:6) and antisense 5′CATGGCCCGGTGTTCCTGCAGTTG3′(bases 927-949; SEQ ID No:7) into the back bone of pAR(DRI) 59/60plasmid digested with EcoRI-BamHI. For the construction ofFLAG-HMK-IL-2Rβ-S⁻ mutant (serine-rich domain is deleted), a plasmidencoding FLAG-HMK-IL-2R WT was digested with Sac-AflII. After fillingboth ends, the plasmid was blunt end ligated. For construction ofFLAG-HMK-IL-2Rβ-A⁻ mutant (acidic domain is deleted), a 1048 bp fragmentobtained from SacI-BamHI digestion of FLAG-HMK-IL-2Rβ-WT was furtherdigested with PstI resulting in 3 fragments of 701, 210 and 136 bp.Fragments 701 and 136 were ligated back into the backbone of SacI-BamHIdigested FLAG-HMK-IL-2Rβ-WT construct. The authenticity of each of theintroduced mutations was confirmed by DNA sequence analysis.

(ii) In FIG. 7a, there are shown schematic representations of the wildtype (WT) and mutant (box 1⁻S⁻; A⁻) IL-2Rβ chain protein constructsprepared as above for expression in COS cells. In FIG. 7b, there areshown schematic representations of the wild type (WTO and mutant (S⁻;A⁻) IL-2Rβ chain protein constructs prepared as above (see also Example1, a(i) and (ii) above) for expression in COS cells. These constructswere introduced into COS cells and bacterial cells and the proteins wereexpressed, affinity purified from lysates of the cells, the purifiedproteins were separated on SDS-PAGE and stained with Commassie blue (forbasic procedures see also Le Clair et al., 1992; Blanar and Rutter,1992). The procedure for expression of the constructs in bacterial cellsfollowed by affinity purification, SDS-PAGE separation and Commassieblue staining has been described above (Example 1, a (i) and (ii)). Theprocedure for expression of the constructs in COS cells followed bySDS-PAGE separation, affinity purification and Commassie staining was asfollows:

COS cells were transfected via the DOTAP method (Boehringer-Mannheim,Indianapolis, Ind.) following the manufacturer's instructions. Thetransfection cocktail contained 5 μg of DNA total and 30 ml of DOTAP ina final volume of 150 ml HBS (25 mM HEPES, pH 7.4 and 100 mM NaCl). TheCOS cells were grown in DMEM medium supplemented with 10%heat-inactivated fetal calf serum, penicillin/streptomycin, 25 mM HEPES,pH 7.4, and L-glutamine. The COS cells were exposed to the transfectioncocktail for 12 hours, washed and subsequently cultured in fresh medium.24 hours after washing approximately 3×10⁶ cells were harvested andwashed twice in chilled PBS. A lysis buffer was prepared and consistedof 150 mM NaCl, 50 mM Tris pH=7.4, 0.5% CHAPS (Pierce), 10% glycerol(Sigma), supplemented with the following protease inhibitors immediatelybefore use: aprotinin (Sigma) 2.5 mg/ml, leupeptin (Boehringer-Mannheim)2.5 mg/ml; Pepstatin A (Boehringer-Mannheim) 2 mg/ml, PMSF (Sigma) 150mg/ml, NaF (Sigma) 100 mM and sodium orthovanadate (Sigma) 1 mM. Thetransfected COS cells were lysed in 0.5 ml of lysis buffer on ice for 10minutes, and subsequently centrifuged at 12,000×g for 5 minutes,remaining supernatants were collected, and supplemented with pre-immuneserum and protein G-agarose beads (BRL-Gibco, Gaithersburg, Md.), whichhad been previously washed in lysis buffer. The samples were incubatedat 4° C. for 30 minutes on a rocker. Supernatants were collected andsupplemented with appropriate antibody, and later the protein G-agarosebeads were added. Samples were washed 3 times for 15 min. each in lysisbuffer and resuspended in Laemmli buffer and subsequently subjected toSDS-PAGE followed by Commassie blue staining for basic procedures (seealso Maslinski, et al., 1992).

Antibodies used in the above affinity purification step (with theprotein G-agarose beads) included: a rabbit anti-serum raised against a14-3-3 protein expressed in bacteria using standard procedures, thisbeing a polyclonal anti-14-3-3 antibody protein; a rabbit anti-human14-3-3 antibody that is cross-reactive with bacterial 14-3-3 proteinspurchased from Upstate Biotechnology; an anti-Raf-1 (C1) antibodypurchased from Santa Cruz Biotechnology; an anti-human IL-2Rβ antibodycalled Mik-ol (as described in Tsudo et al, 1989 and obtained from MTsudo, Kyoto, Japan).

In FIG. 7(c), there is shown a reproduction of the relevant bands of aCommassie blue stained, SDS-PAGE separation of affinity purifiedFLAG-HMK-IL-2Rβ chain related (wild type and mutant) fusion proteinswhich were expressed in the COS cells.

(iii) To determine the nature of the binding of the IL-2Rβ chain toRaf-1 and 14-3-3 proteins, COS cells were transfected, as set forthhereinabove, with constructs encoding full-length or deletional mutantsof the human IL-2Rβ chain, immunoprecipitated with an anti-IL-2Rβ chainantibody Mik-β1 (see (ii) above) and blotted with anti-Raf-1 oranti-14-3-3 antibodies (see (ii) above).

In addition, in order to determine the interaction of the IL-2Rβ chainwith Raf-1 and 14-3-3 proteins in T-cells, lysates of phytohemagglutinin(PHA)-activated peripheral blood mononuclear cells were passed throughanti-FLAG affinity beads containing purified FLAG-HMK-IL-2Rβ relatedproteins (see (i) and (ii) above, as well as Examples 1-3). The absorbedproteins were washed, eluted with FLAG peptide and probed for thepresence of Raf-1 and 14-3-3 proteins on immunoblots. The peripheralblood mononuclear cells were isolated using Ficoll-Hypaque (Pharmacia),stimulated with PHA (Sigma) 5 mg/ml in culture for 72 hours, washed,maintained in culture for 3 days in the presence of IL-2 (Hoffman-LaRoche) 10 U/ml, and then incubated without IL-2 for 24 hours. Washedcells (about 4×10⁷) were lysed in Dounce homogenization buffer,centrifuged (15×10³×g for 15 min.) and supernatants applied onto washedanti-FLAG (M2) affinity column (IBI-Kodak) coated with bacterial lysatesinteracted with one of the FLAG-HMK fusion proteins. After washing with15 ml of buffer containing 50 mM Tris pH=7.4, 150 mM NaCl, proteinsadsorbed onto the anti-FLAG affinity column were eluted with the samebuffer supplemented with FLAG peptide (10⁻⁴ M) and subjected to SDS-PAGEand immunoblotting described hereinabove.

To study the IL-2Rβ chain/Raf-1 interaction in vitro, bacterial lysatescontaining FLAG-HMK-IL-2Rβ chain related and (His)₆ Raf-1 fusionproteins (see also Examples 2, 3 above) were mixed and adsorbed onanti-FLAG beads. The proteins bound on the beads were washed, elutedwith FLAG peptide and probed for the presence of Raf-1 and 14-3-3proteins by immunoblotting.

The results of the above experiments are shown in FIGS. 8a-c:

In FIG. 8a, there is shown a reproduction of immunoblots performed onlysates from transfected COS cells which were transfected with thevarious constructs IL-2Rβ-WT, IL-2Rβ-box 1⁻S⁻, IL-2Rβ-A⁻, or, as acontrol, a vector having no IL-2Rβ construct (vector). The COS celllysates were immunoprecipitated with anti-Raf-1 or anti-14-3-3antibodies. From the results shown in FIG. 8a it is apparent that bothIL-2Rβ-WT and the IL-2Rβ-box 1⁻S⁻ mutant bound both Raf-1 and 14-3-3proteins. In contrast, the IL-2Rβ chain A⁻ mutant failed to bind Raf-1and bound only 14-3-3 proteins.

In FIG. 8b, there is shown a reproduction of an immunoblot performed onlysates from PHA activated peripheral blood mononuclear cells, whichwere passed through anti-FLAG affinity beads containing purifiedFLAG-HMK-IL-2Rβ related proteins. The adsorbed proteins were washed,eluted with FLAG peptide and probed for the presence of Raf-1 and 14-3-3on immunoblots. From the results shown in FIG. 8b, it is apparent thatthe same specific interactions (as in FIG. 8a) also occurred when T-celllysates were passed through IL-2Rβ chain-derived affinity columns, i.e.,IL-2Rβ-WT and the IL-2Rβ-box 1⁻S⁻ mutant but not the IL-2Rβ chain A⁻mutant bound to Raf-1. In these T-cell lysates the Raf-1 protein is atbasal levels as shown by phosphorylation of exogenously added kinaseinactive MEK protein (see Examples 2 and 3 above).

In view of the results shown in FIGS. 8a and 8 b, it was concluded thatthe 70 amino acidic region (A⁻ region) of the IL-2Rβ chain is requiredfor Raf-1 binding, while the 144 amino acid C-terminal portion of theIL-2Rβ chain is required for interaction with 14-3-3 proteins.

(iv) In order to ascertain whether the A⁻ region and C-terminal regionsdirectly bind to Raf-1 and 14-3-3 proteins respectively, a series ofbacterially expressed FLAG-IL-2Rβ chain fusion proteins were tested. Inthese experiments bacterially expressed (His)6 Raf-1 protein was used(see Examples 2 and 3 above, and FIG. 7b for bacterial constructs). Theresults of these experiments are shown in FIG. 8c which is areproduction of an immunoblot performed on bacterial lysates containingFLAG-HMK-IL-2Rβ chain related and (His)6 Raf-1 fusion proteins, whichwere mixed and adsorbed on anti-FLAG beads. The proteins bound to thebeads were washed, eluted with FLAG peptide and probed byimmunoblotting. Since the bacterial lysates contained a 28 kD protein,immunoreactive with antibody raised against a highly conserved region ofthe 14-3-3 protein (residues 119-129 of human 14-3-3) no attempt wasmade to co-express human 14-3-3 proteins. As is apparent from FIG. 8c,there is direct binding between Raf-1 and the acidic region of theIL-2Rβ chain. Further, as in COS cells, 14-3-3 proteins present inbacterial lysates bound directly to the C-terminal portion of the IL-2Rβchain. Thus, it appears that the homology between mammalian andbacterial 14-3-3 proteins is sufficient to preserve the 14-3-3 bindingsite to Raf-1 and the IL-2Rβ chain.

However, it must be also noted that, as arises from FIG. 8c, bacterial14-3-3 bound to the IL-2Rβ chain only in the presence of Raf-1 proteins.Accordingly, it is likely that Raf-1 and 14-3-3 form a complex beforebinding to the A⁻ region (Raf-1) and the C-terminal part (14-3-3) of theIL-2Rβ chain. Once the 14-3-3 protein is bound to IL-2Rβ the requirementto maintain the association with Raf-1 is less stringent as arises fromthe fact that the mutant IL-2Rβ protein lacking the acidic region doesnot bind Raf-1 (FIGS. 8b and c).

The above results therefore suggest that Raf-1 plays a central role inthese three molecular interactions (Raf-1—14-3-3 - IL-2Rβ). This notionis further supported by the observation that the A⁻ region of the IL-2Rβchain is homologous to the effector domain or Ras and Rap1A that bindsto Raf-1 (see, for example, Zhang et al., 1993; Nassar et al., 1995).The homology between Ras (H-Ras) and the A region of IL-2Rβ is depictedschematically in FIG. 9. The interaction between IL-2Rβ chain (aminoacids 371-395) may therefore be a key factor in Raf-1 immobilizationthrough the IL-2Rβ chain at the plasma membrane.

(v) Triggering of the IL-2 receptor complex activates several tyrosinekinases in T cells; these include Jak-1 (Miyazaki et al., 1994; Jak-3(see, for example, Johnstein et al., 1994) and p56^(lck) (Minami et al.,1995). Previously, we showed that tyrosine kinase dependent dissociationof Raf-1 from the IL-2Rβ chain is a prerequisite for Raf-1 activation byIL-2 (Maslinski et al., 1992). Although both Jak-1 and p56^(lck) arebound to the non-activated IL-2Rβ chain, the observation that p56^(lck)also binds to the A⁻ region (Minami et al., 1993) prompted us to examineits role in the dissociation of Raf-1/14-3-3 proteins from the IL-2Rβchain. In order to perform this examination, COS cells were transfectedwith IL-2Rβ chain alone, or co-transfected with lck or fyn, then lysedwith anti-IL-2Rβ chain antibody. The immunoprecipitates were then probedfor the presence of Raf-1 and 14-3-3 proteins by immunoblotting (allprocedures as detailed hereinabove).

The results of this examination are shown in FIG. 10a which is areproduction of the above immunoblot. From these results it is apparentthat co-transfection of COS cells with the IL-2Rβ chain and p56^(lck)resulted in the abrogation of Raf-1/14-3-3 binding to the IL-2Rb chain.In contrast, another src-like kinase, p59^(fyn) did not cause thisdissociation. In addition, a study of the dissociation of pre-formedIL-2Rβ chain/Raf-1/14-3-3 complexes by enzymatically active p56^(lck)was also carried out. Pre-formed IL-2Rβ chain/(His)₆ Raf-1/bacterial14-3-3 complexes were prepared (see above in respect of FIGS. a-c),exposed to catalytically active p56^(lck) (Upstate Biotechnology),washed and eluted with FLAG peptide. Eluates were separated on SDS-PAGEand tested for the presence of Raf-1 proteins. The results of this studyare shown in FIG. 10b which is a reproduction of an SDS-PAGE gel inwhich is depicted the Raf-1 bands. From these results it is apparentthat dissociation of pre-formed IL-2Rβ chain/Raf-1/14-3-3 complexes byenzymatically active p56^(lck) could also be seen in vitro. It wastherefore concluded that activation of p56^(lck) contributes to thedissociation of Raf-1/14-3-3 proteins from the IL-2Rβ chain during IL-2mediated Raf-1 activation. Since there is no indication that p56^(lck)interacts with Ras-like sequence of the IL-2Rβ chain, it seems thatbinding of Raf-1 and p56^(lck) to distinct subdomains of the sameA-region, co-localize kinase and its substrate for fast enzymaticreaction occurring during IL-2R activation.

Taken together, the above results show that Raf-1/14-3-3 complexesdirectly associate with the IL-2Rβ chain: the A-region of the receptoris required for Raf-1 binding while the C-terminal portion of themolecule interacts with 14-3-3. These results are consistent with (i)the homology between acidic domain of the IL-2Rβ chain and the effectordomain of Ras and Rap1A that binds Raf-1 (FIG. 9); and (ii) theexistence of pre-formed Raf-1/14-3-3 protein complexes in the cytosol orco-localized to the plasma membrane (see also Fanti et al., 1994). TheIL-2Rβ chain may therefore bypass the requirement for Ras activation inthe membrane localization of Raf-1 (see Leevers et al., 1994; Stokoe etal., 1994). Two distinct regions of the IL-2Rβ chain involved in theoptimal binding of Raf-1 and 14-3-3 proteins (the acidic A andC-terminal regions, respectively) may enable “permissive” Raf-1 bindingand activation, i.e., the IL-2Rβ chain mutant lacking A-region may bindsome of Raf-1 proteins through the binding to 14-3-3 proteins associatedwith C-terminal part (14-3-3 binding domain) of the receptor. Forexample, BAF cells expressing the mutant IL-2Rβ chain lacking theA-region still respond to IL-2 albeit more weakly than those expressingthe wild-type molecule (Hatakeyama et al., 1989). Alternatively, Raf-1activation occurring in the absence of the IL-2Rβ A domain may beachieved via IL-2 induced activation of Ras (see for example,Izquierdo-Pastor et al., 1995).

In view of the results set forth hereinabove in Examples 1-4, it may beconcluded that in accordance with the present invention, it has beenshown that the IL-2Rβ chain and Raf-1 interact directly and that theIL-2Rβ chain and 14-3-3 proteins also interact directly. Further, Raf-1and 14-3-3 proteins bind at different sites on the IL-2Rβ chain and formcomplexes. The portion of the intracellular domain of the IL-2Rβ chainthat is required for binding to Raf-1 has now been defined, this beingthe so-called acidic region encompassing amino acid residues 313-382 ofthe mature human IL-2Rβ chain (see also Example 6 below and FIGS. 11 and12). Further, it has now also been shown that the same portion of theIL-2Rβ chain (acidic domain) is needed for activation of the Raf-1enzymatic activity (the so-called protein kinase activity). Moreover,while the above acidic domain of the IL-2Rβ chain, that is homologous tothe Ras effector domain, is critical for Raf-1 binding, it is theC-terminal portion of the receptor which interacts with 14-3-3 proteins.In the presence of enzymatically active p56^(lck) but not p59^(fyn),Raf-1/14-3-3 complexes dissociate from the IL-2Rβ chain, an eventdirectly related to IL-2 mediated activation of IL-2R and subsequentintracellular signalling. Two in vitro binding assays have beendeveloped which are suitable for screening a number of samples for thepresence of compounds or substances which have blocking activity, i.e.,that are capable of blocking the binding or interaction of the IL-2Rβchain to Raf-1, and thereby blocking the signaling pathway initiated byIL-2/IL-15 binding to its receptor (see Examples 5 and 6 below). Suchcompounds or substances would thereby be useful for the treatment ofautoimmune diseases in general, transplant rejection andgraft-versus-host rejection process in particular, by being able toblock the IL-2/IL-15-mediated signaling pathway.

EXAMPLE 5 In Vitro Assays for Testing Compounds Capable of Disruting theIL-2R Signaling Pathway

As set forth in Examples 1-4 above, two in vitro assays have beendeveloped in accordance with the present invention. The first such assayis a cell-free system in which bacterially produced or mammalian cell(COS cells) produced IL-2Rβ chain fusion proteins are interacted with Tcell lysates to isolate, identify and characterize compounds, forexample, Raf-1 protein, and 14-3-3 proteins capable of bindingspecifically to the IL-2Rβ chain intracellular domain or portionsthereof. The second such assay is the so-called totally cell-free systemin which bacterially produced or mammalian cell produced IL-2Rβ chainfusion proteins are interacted with bacterially produced Raf-1 protein((His)₆-Raf-1) and 14-3-3 proteins to isolate, identify and characterizethe nature of the binding between the IL-2Rβ chain intracellular domainor portions thereof and the Raf-1 and 14-3-3 proteins. In both of theseassays it is possible to determine both qualitatively and quantitativelythe extent of binding between the IL-2Rβ chain intracellular domain orportions thereof, and Raf-1 and 14-3-3 proteins. In the cell-free systemit is also possible to determine protein kinase reaction which occursfollowing the binding of Raf-1 and 14-3-3 proteins to a specific regionof the intracellular domain of IL-2Rβ (the acidic domain and the acidicand proline-rich C-terminal region). This determination of the proteinkinase reaction is an indicator of the initiation of the intracellularsignaling process which is apparently initiated by the binding of Raf-1and/or 14-3-3 to IL-2Rβ. Therefore, the determination of the proteinkinase activity in vitro provides a reliable assay means for determiningwhether or not another compound, for example, peptides, organiccompounds, etc., are capable of disrupting the binding between Raf-1 and14-3-3 proteins and IL2-Rβ and thereby inhibiting the kinase reactionwhich is essential to the intracellular signaling mediated by IL-2R.

In the totally cell-free system it arises that in order to be able todetermine the Raf-1 protein kinase reaction an additional factor(s) isrequired, this being most likely a protein of the 14-3-3 family. Theestablishment of this totally cell-free system and its success formeasuring the interaction, i.e., binding between Raf-1, 14-3-3 proteinsand IL-2Rβ, permits the further development of this system, i.e., usethereof to isolate and identify the additional factor(s) necessary forutilization of the system to determine the protein kinase activityfollowing binding of Raf-1 and 14-3-3 to IL-2Rβ.

In order to screen compounds such as peptides, organic molecules, etc.,for their ability to bind to either the IL-2Rβ chain intracellulardomain or specific essential regions thereof and thereby causeinhibition of binding of IL-2Rβ to Raf-1 and 14-3-3 it is possible toutilize any of the above in vitro assay systems. In such a screeningassay, bacterially produced or mammalian cell (COS cells) producedIL-2Rβ chain intracellular domain (WT) and/or IL-2Rβ chain intracellulardomain analogs such as those containing only the acidic domain orcontaining both the acidic and proline-rich C terminal domains may beemployed as the substrate to which will be exposed samples containingthe peptides, organic compounds, etc., which are to be screened toisolate those which specifically bind the IL-2Rβ chain. Once suchcompounds are obtained, they may be further tested in these screeningassays for their ability to inhibit Raf-1 and/or 14-3-3 binding and/orthe resulting protein kinase reaction. The procedures to be used inthese assays are as detailed hereinabove in Examples 1-4.

It should be mentioned that in the above screening assays it is possibleto readily develop an ELISA-type assay system by binding of the FLAGantibody to a microtiter plate sequentially followed bybacterially-expressed or mammalian cell-expressed IL-2Rβ chain-FLAGfusion protein and bacterially-expressed or mammalian cell-expressedRaf-1 and 14-3-3 proteins in the presence (or absence=control) of apotential inhibitor to be screened and finally by an antibody to Raf-1and/or 14-3-3, this antibody being labelled by standard labels, e.g.,radioactive, fluorescent labels or coupled to an enzyme which generatesa colored product in the presence of its substrate.

EXAMPLE 6 Compounds Capable of Binding to the Acidic Domain of theIL-2Rβ Intracellular Domain That are Able to Inhibit the Binding ofRaf-1 and/or 14-3-3 Proteins to the IL-2Rβ Chain

As set forth in Examples 1-4 above, the acidic region of the IL-2Rβchain is the region responsible for direct binding to Raf-1 and theC-terminal region is responsible for direct binding to 14-3-3 proteins.The acidic region encompasses amino acids 313-382 of the mature humanIL-2Rβ chain. Raf-1 and 14-3-3 also form complexes and appear to bindIL-2Rβ and to dissociate therefrom in the form of complexes.

The proline-rich C-terminal portion of the IL-2Rβ chain (amino acids383-525) is not critical for Raf-1 binding, but is critical for 14-3-3binding; this portion of the IL-2Rβ chain may at most stabilize Raf-1binding via the binding of 14-3-3 at this region which is complexed toRaf-1. In FIG. 11, there is shown a scheme of the essential portions ofthe IL-2Rβ intracellular domain (intracytoplasmic region) that areinvolved in binding to Raf-1 and are thus directly involved in the IL-2Rmediated intracellular signaling. In FIG. 12, there is shown,schematically, the amino acid sequence of the human IL-2Rβ chain. InFIG. 12, the extra cytoplasmic domain is in the upper part of the figure(capital letters); the peptide leader region is indicated by lowerletters and the transmembrane region is indicated by underlined letters;and the intracytoplasmic domain is shown in the lower part of thefigure, in which the acidic region (a.a. 313-382) is indicated by dottedunderlined letters within which region (a.a. 345-371) are shown byitalic capital letters the amino acid residues involved directly inIL-2Rβ interaction, of which residues those shown by bold capital italicletters are the acidic residues. The serine residues of the serine-richregion in the intracytoplasmic domain are indicated by crossed-outcapital S letters.

One such peptide which is likely to be capable of disrupting the bindingbetween Raf-1 and IL-2Rβ and between Raf-1/14-3-3 and IL-2Rβ is a 27amino acid peptide derived from analysis of deletion mutants (seeExamples 1-4 above), being part of the acidic domain and having asequence corresponding to amino acids 345-371 of the mature IL-2Rβ chainprotein (i.e., peptide having amino acid residues corresponding to aminoacids 370 to 396 of SEQ ID No:2, see FIG. 12).

Analogs of the above 27 amino acid peptide will be made by standardchemical synthesis procedures well known in the art or by standardrecombinant DNA techniques. Such analogs will include those having oneor more amino acids deleted, added or replaced with respect to above 27amino acid peptide and which will be characterized by their ability toinhibit the binding between Raf-1 and/or 14-3-3 proteins and IL-2Rβ.

Other proteins or peptides which are likely to be capable ofspecifically binding to Raf-1 and/or IL-2Rβ and which may be capable ofinhibiting the binding between Raf-1 and/or 14-3-3 proteins and IL-2Rβinclude one or more proteins derived from the 14-3-3 family of proteinsor specific peptides derived therefrom or any analogs, derivativesthereof.

As mentioned in Example 4 above, other proteins, peptides, organiccompounds, etc., which are capable of binding specifically to Raf-1and/or 14-3-3 proteins or IL-2Rβ chain intracellular domain and therebyinhibit the binding of Raf-1 and/or 14-3-3 proteins to IL-2Rβ, may bereadily obtained by utilization of the in vitro screening assays.

It should be mentioned that of the compounds of potential Raf-1/IL-2Rβor Raf-1/14-3-3/IL-2Rβ binding inhibitory capability to be screened,organic compounds with some lipophilic characteristics may be mostuseful in view of the fact that in practice, such compounds to be usedpharmaceutically would have to have the ability to pass through the cellmembrane. For instance, peptides can be chemically modified orderivatized to enhance their permeability across the cell membrane andfacilitate the transport of such peptides through the membrane and intothe cytoplasm. Muranishi et al. (1991) reported derivatizingthyrotropin-releasing hormone with lauric acid to form a lipophiliclauroyl derivative with good penetration characteristics across cellmembranes. Zacharia et al. (1991) also reported the oxidation ofmethionine to sulfoxide and the replacement of the peptide bond with itsketomethylene isoester (COCH₂) to facilitate transport of peptidesthrough the cell membrane. These are just some of the knownmodifications and derivatives that are well within the skill of those inthe art.

Furthermore, the compounds of the present invention, which are capableof inhibiting the binding of Raf-1 and/or 14-3-3 proteins to thecytoplasmic domain of IL-2Rβ, can be conjugated or complexed withmolecules that facilitate entry into the cell.

U.S. Pat. No. 5,149,782 discloses conjugating a molecule to betransported across the cell membrane with a membrane blending agent suchas fusogenic polypeptides, ion-channel forming polypeptides, othermembrane polypeptides, and long chain fatty acids, e.g., myristic acid,palmitic acid. These membranes blending agents insert the molecularconjugates into the lipid bilayer of cellular membranes and facilitatetheir entry into the cytoplasm.

Low et al., U.S. Pat. No. 5,108,921, reviews available methods fortransmembrane delivery of molecules such as, but not limited to,proteins and nucleic acids by the mechanism of receptor mediatedendocytotic activity. These receptor systems include those recognizinggalactose, mannose, mannose 6-phosphate, transferrin,asialoglycoprotein, transcobalamin (vitamin B₁₂), α-2 macroglobulins,insulin and other peptide growth factors such as epidermal growth factor(EGF). Low et al. teaches that nutrient receptors, such as receptors forbiotin and folate, can be advantageously used to enhance transportacross the cell membrane due to the location and multiplicity of biotinand folate receptors on the membrane surfaces of most cells and theassociated receptor mediated transmembrane transport processes. Thus, acomplex formed between a compound to be delivered into the cytoplasm anda ligand, such as biotin or folate, is contacted with a cell membranebearing biotin or folate receptors to initiate the receptor mediatedtrans-membrane transport mechanism and thereby permit entry of thedesired compound into the cell.

Further, screening directed at small peptides, e.g., that noted above(having between 20-30 amino acid), is also advantageous to isolate anddevelop more stable peptidomimetic-type drugs. Once such compounds,peptides, etc., have been screened and found to be capable of binding toRaf-1 and/or 14-3-3 or IL-2Rβ and thereby block the binding betweenthese proteins, these compounds will then be assessed for their expectedutility in inhibition of autoimmune diseases in general, and forprevention of transplantation rejection in particular.

The above noted peptides in accordance with the invention may be anypeptide of natural origin isolated in the above in vitro screeningassays or may be any peptide produced by standard peptide synthesisprocedures. Suitable peptides are those capable of interfering with theinteraction between Raf-1 and/or 14-3-3 proteins with IL-2Rβ and therebyinhibiting the intracellular signalling process mediated by IL-2Rβ.

Likewise, the above noted organic compounds in accordance with thepresent invention may be any known pharmaceutically utilized compound orany newly synthetized compound prepared by standard chemical synthesismethods. Suitable such compounds are those capable of interfering withthe interaction between Raf-1 and/or 14-3-3 proteins with IL-1 2Rβ andthereby inhibiting the intracellular signalling process mediated byIL-2Rβ.

The above peptides, organic compounds, etc., of the invention may thusbe used as the active ingredients in pharmaceutical compositions for thetreatment of autoimmune diseases in general, or host-versus-graftreactions in particular. Hence the pharmaceutical compositions of theinvention are those comprising a pharmaceutically acceptable carrier,stabilizer or excipient and the above active ingredients of theinvention.

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration. Any accepted mode ofadministration can be used and determined by those skilled in the art.For example, administration may be by various parenteral routes such assubcutaneous, intravenous, intradermal, intramuscular, intraperitoneal,intranasal, transdermal, or buccal routes. Parenteral administration canbe by bolus injection or by gradual perfusion over time.

It is understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art.

The total dose required for each treatment may be administered bymultiple doses or in a single dose. The pharmaceutical composition ofthe present invention may be administered alone or in conjunction withother therapeutics directed to the condition, or directed to othersymptoms of the condition.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions, which may containauxiliary agents or excipients which are known in the art, and can beprepared according to routine methods.

Pharmaceutical compositions comprising the inhibitory compounds of thepresent invention include all compositions wherein the inhibitorycompound is contained in an amount effective to achieve its intendedpurpose. In addition, the pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspension of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions that may contain substances which increasethe viscosity of the suspension include, for example, sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. optionally, thesuspension may also contain stabilizers.

Pharmaceutical compositions include suitable solutions foradministration by injection, and contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active component (i.e.,compounds that inhibit the binding of Raf-1 or 14-3-3 proteins toIL-2Rβ) together with the excipient. Compositions which can beadministered rectally include suppositories.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional method steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

1. Giri et al., EMBO J., 13:2822 (1984).

2. Bamford et al., PNAS, 91:4940 (1994).

3. Smith, K. A., Science, 240:1169 (1988).

4. Waldmann, T. A., “The IL-2/IL-2 receptor system: A target forrational immune intervention”, Immunol. Today, 14:264 (1993).

5. Robb, R. J., et al., J. Exa. Med., 160:1126 (1984).

6. Siegal, J. P., et al., Science, 238:75 (1987).

7. Hatakeyama, M., et al., “A restricted cytoplasmic region of IL-2receptor β chain is essential for growth signal transduction but not forligand binding and internalization”, Cell, 59:837 (1989).

8. Nakamura, Y., et al., “Heterodimerization of the IL-2 receptor β- andgamma-chain cytoplasmic domains is required for signaling”, Nature,369:330 (1994).

9. Noguchi, M., et al., “Interleukin-2 receptor gamma chain: afunctional component of the Interleukin-7 receptor”, Science, 262:1877(1993).

10. Russell, S. M., et al., “Interleukin-2 receptor gamma chain: afunctional component of the Interleukin-4 receptor”, Science, 262:1880(1993).

11. Michiel, D. F., et al., “Regulation of the interleukin 2 receptorcomplex tyrosine kinase activity in vitro”, Cytokine, 3:428 (1991).

12. Remillard, B., et al., (1991), “Interleukin-2 receptor regulatesactivation of phosphatidylinositol 3 kinase”, J. Biol. Chem., 266:14167.

13. Benedict, S. H., et al., (1987), J. Immunol., 139:1694.

14. Ferris, D. K., et al., (1989), J. Immunol., 143:870.

15. Saltzman, E. M., et al., (1988), J. Biol. Chem., 263:6956.

16. Asao, H., et al., (1990), J. Exp. Med., 171:637.

17. Mills, G. B., et al., (1990), J. Biol. Chem., 265:3561.

18. Merida, I. and G. N. Gaulton, (1990), J. Biol. Chem., 265:3561.

19. Fung, M. R., et al., (1991), “A tyrosine kinase physicallyassociates with the β-subunit of the human IL-2 receptor”, J. Immunol.,147:1253.

20. Hatakeyama, M., et al., (1991), Science, 252:1523.

21. Turner, B., et al., (1991), “Interleukin-2 induces tyrosinephosphorylation and activation of p72-p74 Raf-1 kinase in a T-cellline”, PNAS (USA), 88:1227.

22. Valentine, M. V., et al., (1991), Eur. J. Immunol., 21:913.

23. Carroll, M. P., et al., (1990), J. Biol. Chem., 265:19812.

24. Morrison, D. K., et al., (1988), PNAS (USA), 85:8855.

25. Baccarini, M., et al., (1991), J. Biol. Chem., 266:10941.

26. Kovacina, K. S., et al., (1990), J. Biol. Chem., 265:12115.

27. Blackshear, P. J., et al., (1990), J. Biol. Chem., 265:12131.

28. App, H., et al., (1991), Mol. Cell Biol., 11:913.

29. Heidecker, G., et al., (1992), “The role of raf-1 phosphorylation insignal transduction”, Adv. Cancer Res., 58:53.

30. Carroll, M. P., et al., (1991), J. Biol. Chem., 266:14964.

31. D'Andrea, A. D., et al., (1989), Cell, 58:1023.

32. Zmuidzinas, A., et al., (1991), “Interleukin-2 triggered Raf-1expression, phosphorylation, and associated kinase activity increasethrough G1 and S in CD3-stimulated primary human T cells”, Mol. CellBiol., 11:2794.

33. Maslinski, W., B. et al., (1992), “Interleukin-2 (IL-2) inducestyrosine kinase-dependent translocation of active raf-1 from the IL-2receptor into the cytosol”, J. Biol. Chem., 267:15281.

34. Downward, J., et al., (1990), “Stimulation of p21ras upon T-cellactivation”, Nature, 346:719.

35. Williamson, P., et al. (1994), “The membrane proximal segment of theIL-2 receptor β-chain acidic region is essential for IL2-dependentprotein tyrosine kinase activation”, Leukemia 8 Suppl., 1:S186.

36. Nelson, B. H., et al., (1994), “Cytoplasmic domains of theinterleukin-2 receptor β and γ chains mediate the signal for T-cellproliferation”, Nature, 369:333.

37. Schorle, H., et al., (1991), “Development and function of T cells inmice rendered interleukin-2-deficient by gene targeting”, Nature,352:621.

38. Kundig, T. M., et al., (1993), “Immune responses ininterleukin-2-deficient mice”, Science, 262:1059.

39. Noguchi, M., et al., (1993), Cell, 73:147.

40. Strom, T. B., et al., (1993), “Interleukin-2 receptor-directedtherapies: Antibody- or cytokine-based targeting molecules”, Ann. Rev.Med., 44:343.

41. Riedel, D., et al., (1993), “The mitogenic response of T cells tointerleukin-2 requires Raf-1”, Eur. J. Immunol., 23:3146.

42. Freed, E., et al., (1994), “Binding of 14-3-3 proteins to theprotein kinase raf and effects on its activation”, Science, 265:1713.

43. Irie, K., et al., (1994), “Stimulatory effects of yeast andmammalian 14-3-3 proteins on the Raf protein kinase”, Science, 265:1716.

44. LeClair, K. P., et al., (1992), PNAS (USA), 89:8145-8149.

45. Blanar, M. A. and Rutter, R. J., (1992), Science, 256:1014-1018.

46. McGrew, B. R. et al., (1992), Oncogene, 7:33-42.

47. Bruder, J. T., et al., (1992), Genes and Development, 6:545-556.

48. Stanton, V. P., et al., (1989), Mol. Cell Biol., 9:639-647.

49. Murakami, M. et al., (1991), PNAS (USA), 88:11349-11353.

50. Tsudo M. et al., (1989), PNAS (USA), 86:1982-1986.

51. Zhang X. et al., (1993), Nature, 364:308-313.

52. Nassar N. et al., (1995), Nature, 375:554-560.

53. Miyazaki T. et al., (1994), Science, 266:1045-1047.

54. Johnston, J. A. et al., (1994), Nature, 370:151-153.

55. Minami Y. et al., (1995), Immunity, 2:89-100.

56. Minami Y. et al., (1993), EMBO J., 12:759-768.

57. Fanti, W. J. et al., (1994), Nature, 371:612-614.

58. Leevers, S. J. et al., (1994), Nature, 369:411-414.

59. Stokoe D. et al., (1994), Science, 264:1463-1467.

60. Izquierdo-Pastor et al., (1995), Immunoloqy Today, 16:159-164.

61. Moore, B. E. and Perez, V. J., (1967), In: Physiological andBiochemical Aspects of Nervous Integration, F. D. Carlson, Eds.,Prentice-Hall, Englewood Cliffs, N.J.

64. Aitken, A. et al., (1992), Trends Biochem. Sci. 17:498.

63. Fu, H. et al., (1994), Science 266:126-129.

64. Reuther, G. W. et al., (1994), Science 266:129.

65. Pallas, D. C. et al., (1994), Science 265:535.

66. Morrison, D., (1994), Science 266:56-57.

67. Muranishi, S. et al., (1991), Pharm. Research 8:649.

68. Zaccharia, S. et al. (1991), Eur. J. Pharmacol. 203:353-357.

7 1 1656 DNA Homo sapiens CDS (1)..(1653) 1 atg gcg gcc cct gct ctg tcctgg cgt ctg ccc ctc ctc atc ctc ctc 48 Met Ala Ala Pro Ala Leu Ser TrpArg Leu Pro Leu Leu Ile Leu Leu 1 5 10 15 ctg ccc ctg gct acc tct tgggca tct gca gcg gtg aat ggc act tcc 96 Leu Pro Leu Ala Thr Ser Trp AlaSer Ala Ala Val Asn Gly Thr Ser 20 25 30 cag ttc aca tgc ttc tac aac tcgaga gcc aac atc tcc tgt gtc tgg 144 Gln Phe Thr Cys Phe Tyr Asn Ser ArgAla Asn Ile Ser Cys Val Trp 35 40 45 agc caa gat ggg gct ctg cag gac acttcc tgc caa gtc cat gcc tgg 192 Ser Gln Asp Gly Ala Leu Gln Asp Thr SerCys Gln Val His Ala Trp 50 55 60 ccg gac aga cgg cgg tgg aac caa acc tgtgag ctg ctc ccc gtg agt 240 Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys GluLeu Leu Pro Val Ser 65 70 75 80 caa gca tcc tgg gcc tgc aac ctg atc ctcgga gcc cca gat tct cag 288 Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu GlyAla Pro Asp Ser Gln 85 90 95 aaa ctg acc aca gtt gac atc gtc acc ctg agggtg ctg tgc cgt gag 336 Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg ValLeu Cys Arg Glu 100 105 110 ggg gtg cga tgg agg gtg atg gcc atc cag gacttc aag ccc ttt gag 384 Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp PheLys Pro Phe Glu 115 120 125 aac ctt cgc ctg atg gcc ccc atc tcc ctc caagtt gtc cac gtg gag 432 Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln ValVal His Val Glu 130 135 140 acc cac aga tgc aac ata agc tgg gaa atc tcccaa gcc tcc cac tac 480 Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser GlnAla Ser His Tyr 145 150 155 160 ttt gaa aga cac ctg gag ttc gag gcc cggacg ctg tcc cca ggc cac 528 Phe Glu Arg His Leu Glu Phe Glu Ala Arg ThrLeu Ser Pro Gly His 165 170 175 acc tgg gag gag gcc ccc ctg ctg act ctcaag cag aag cag gaa tgg 576 Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu LysGln Lys Gln Glu Trp 180 185 190 atc tgc ctg gag acg ctc acc cca gac acccag tat gag ttt cag gtg 624 Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr GlnTyr Glu Phe Gln Val 195 200 205 cgg gtc aag cct ctg caa ggc gag ttc acgacc tgg agc ccc tgg agc 672 Arg Val Lys Pro Leu Gln Gly Glu Phe Thr ThrTrp Ser Pro Trp Ser 210 215 220 cag ccc ctg gcc ttc agg aca aag cct gcagcc ctt ggg aag gac acc 720 Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala AlaLeu Gly Lys Asp Thr 225 230 235 240 att ccg tgg ctc ggc cac ctc ctc gtgggc ctc agc ggg gct ttt ggc 768 Ile Pro Trp Leu Gly His Leu Leu Val GlyLeu Ser Gly Ala Phe Gly 245 250 255 ttc atc atc tta gtg tac ttg ctg atcaac tgc agg aac acc ggg cca 816 Phe Ile Ile Leu Val Tyr Leu Leu Ile AsnCys Arg Asn Thr Gly Pro 260 265 270 tgg ctg aag aag gtc ctg aag tgt aacacc cca gac ccc tcg aag ttc 864 Trp Leu Lys Lys Val Leu Lys Cys Asn ThrPro Asp Pro Ser Lys Phe 275 280 285 ttt tcc cag ctg agc tca gag cat ggagga gac gtc cag aag tgg ctc 912 Phe Ser Gln Leu Ser Ser Glu His Gly GlyAsp Val Gln Lys Trp Leu 290 295 300 tct tcg ccc ttc ccc tca tcg tcc ttcagc cct ggc ggc ctg gca cct 960 Ser Ser Pro Phe Pro Ser Ser Ser Phe SerPro Gly Gly Leu Ala Pro 305 310 315 320 gag atc tcg cca cta gaa gtg ctggag agg gac aag gtg acg cag ctg 1008 Glu Ile Ser Pro Leu Glu Val Leu GluArg Asp Lys Val Thr Gln Leu 325 330 335 ctc ctg cag cag gac aag gtg cctgag ccc gca tcc tta agc agc aac 1056 Leu Leu Gln Gln Asp Lys Val Pro GluPro Ala Ser Leu Ser Ser Asn 340 345 350 cac tcg ctg acc agc tgc ttc accaac cag ggt tac ttc ttc ttc cac 1104 His Ser Leu Thr Ser Cys Phe Thr AsnGln Gly Tyr Phe Phe Phe His 355 360 365 ctc ccg gat gcc ttg gag ata gaggcc tgc cag gtg tac ttt act tac 1152 Leu Pro Asp Ala Leu Glu Ile Glu AlaCys Gln Val Tyr Phe Thr Tyr 370 375 380 gac ccc tac tca gag gaa gac cctgat gag ggt gtg gcc ggg gca ccc 1200 Asp Pro Tyr Ser Glu Glu Asp Pro AspGlu Gly Val Ala Gly Ala Pro 385 390 395 400 aca ggg tct tcc ccc caa cccctg cag cct ctg tca ggg gag gac gac 1248 Thr Gly Ser Ser Pro Gln Pro LeuGln Pro Leu Ser Gly Glu Asp Asp 405 410 415 gcc tac tgc acc ttc ccc tccagg gat gac ctg ctg ctc ttc tcc ccc 1296 Ala Tyr Cys Thr Phe Pro Ser ArgAsp Asp Leu Leu Leu Phe Ser Pro 420 425 430 agt ctc ctc ggt ggc ccc agcccc cca agc act gcc cct ggg ggc agt 1344 Ser Leu Leu Gly Gly Pro Ser ProPro Ser Thr Ala Pro Gly Gly Ser 435 440 445 ggg gcc ggt gaa gag agg atgccc cct tct ttg caa gaa aga gtc ccc 1392 Gly Ala Gly Glu Glu Arg Met ProPro Ser Leu Gln Glu Arg Val Pro 450 455 460 aga gac tgg gac ccc cag cccctg ggg cct ccc acc cca gga gtc cca 1440 Arg Asp Trp Asp Pro Gln Pro LeuGly Pro Pro Thr Pro Gly Val Pro 465 470 475 480 gac ctg gtg gat ttt cagcca ccc cct gag ctg gtg ctg cga gag gct 1488 Asp Leu Val Asp Phe Gln ProPro Pro Glu Leu Val Leu Arg Glu Ala 485 490 495 ggg gag gag gtc cct gacgct ggc ccc agg gag gga gtc agt ttc ccc 1536 Gly Glu Glu Val Pro Asp AlaGly Pro Arg Glu Gly Val Ser Phe Pro 500 505 510 tgg tcc agg cct cct gggcag ggg gag ttc agg gcc ctt aat gct cgc 1584 Trp Ser Arg Pro Pro Gly GlnGly Glu Phe Arg Ala Leu Asn Ala Arg 515 520 525 ctg ccc ctg aac act gatgcc tac ttg tcc ctc caa gaa ctc cag ggt 1632 Leu Pro Leu Asn Thr Asp AlaTyr Leu Ser Leu Gln Glu Leu Gln Gly 530 535 540 cag gac cca act cac ttggtg tag 1656 Gln Asp Pro Thr His Leu Val 545 550 2 551 PRT Homo sapiens2 Met Ala Ala Pro Ala Leu Ser Trp Arg Leu Pro Leu Leu Ile Leu Leu 1 5 1015 Leu Pro Leu Ala Thr Ser Trp Ala Ser Ala Ala Val Asn Gly Thr Ser 20 2530 Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp 35 4045 Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp 50 5560 Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser 65 7075 80 Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln 8590 95 Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu100 105 110 Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro PheGlu 115 120 125 Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val HisVal Glu 130 135 140 Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln AlaSer His Tyr 145 150 155 160 Phe Glu Arg His Leu Glu Phe Glu Ala Arg ThrLeu Ser Pro Gly His 165 170 175 Thr Trp Glu Glu Ala Pro Leu Leu Thr LeuLys Gln Lys Gln Glu Trp 180 185 190 Ile Cys Leu Glu Thr Leu Thr Pro AspThr Gln Tyr Glu Phe Gln Val 195 200 205 Arg Val Lys Pro Leu Gln Gly GluPhe Thr Thr Trp Ser Pro Trp Ser 210 215 220 Gln Pro Leu Ala Phe Arg ThrLys Pro Ala Ala Leu Gly Lys Asp Thr 225 230 235 240 Ile Pro Trp Leu GlyHis Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 245 250 255 Phe Ile Ile LeuVal Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro 260 265 270 Trp Leu LysLys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe 275 280 285 Phe SerGln Leu Ser Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu 290 295 300 SerSer Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro 305 310 315320 Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu 325330 335 Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn340 345 350 His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe PheHis 355 360 365 Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr PheThr Tyr 370 375 380 Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val AlaGly Ala Pro 385 390 395 400 Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro LeuSer Gly Glu Asp Asp 405 410 415 Ala Tyr Cys Thr Phe Pro Ser Arg Asp AspLeu Leu Leu Phe Ser Pro 420 425 430 Ser Leu Leu Gly Gly Pro Ser Pro ProSer Thr Ala Pro Gly Gly Ser 435 440 445 Gly Ala Gly Glu Glu Arg Met ProPro Ser Leu Gln Glu Arg Val Pro 450 455 460 Arg Asp Trp Asp Pro Gln ProLeu Gly Pro Pro Thr Pro Gly Val Pro 465 470 475 480 Asp Leu Val Asp PheGln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala 485 490 495 Gly Glu Glu ValPro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro 500 505 510 Trp Ser ArgPro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg 515 520 525 Leu ProLeu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly 530 535 540 GlnAsp Pro Thr His Leu Val 545 550 3 25 PRT Homo sapiens 3 Thr Ile Gln LeuIle Gln Asn His Phe Val Asp Glu Tyr Asp Pro Thr 1 5 10 15 Ile Glu AspSer Tyr Arg Lys Gln Val 20 25 4 17 DNA Sense oligonucleotide 4catggctgaa gaaggtc 17 5 17 DNA Antisense oligonucleotide 5 ttaagaccttcttcagc 17 6 24 DNA Sense oligonucleotide 6 aattcaactg caggaacacc gggc24 7 24 DNA Antisense oligonucleotide 7 catggcccgg tgttcctgca gttg 24

What is claimed is:
 1. A compound capable of disrupting the binding ofRaf-1 protein and 14-3-3 to IL-2Rβ and selected from the groupconsisting of a peptide having the amino acid sequence corresponding toresidues 370 to 396 of SEQ ID NO:2, an analog of said peptide where oneamino acid residue of residues 370 to 396 of SEQ ID NO:2 is replacedwith a different amino acid residue or deleted, and a chemicalderivative of said peptide which enhances the peptide's permeabilityacross the cell membrane or facilitates the transport of the Peptidethrough the membrane into the cytoplasm, wherein said compound is not afull length human IL-2Rβ chain.
 2. A composition comprising: a compoundaccording to claim 1 or a mixture of two or more different compoundsaccording to claim 1; and a pharmaceutically acceptable carrier,excipient or diluent.